Effective Retrovirus-Mediated Gene Transfer in Normal and Mutant Human Melanocytes

Abstract

Melanocytes represent the second most important cell type in the skin and are primarily responsible for the pigmentation of skin, hair, and eyes. Their function may be affected in a number of inherited and acquired disorders, characterized by hyperpigmentation or hypopigmentation, consequent aesthetic problems, and increased susceptibility to sun-mediated skin damage and photocarcinogenesis. Nevertheless, the possibility of genetically manipulating human melanocytes has been hampered so far by a number of limitations, including their resistance to retroviral infection. To address the problem of human melanocyte transduction, we generated a melanocyte culture from a patient affected with ocular albinism type 1 (OA1), an X-linked pigmentation disorder, characterized by severe reduction of visual acuity, retinal hypopigmentation, and the presence of macromelanosomes in skin melanocytes and retinal pigment epithelium (RPE). The cultured patient melanocytes displayed a significant impairment in replication ability and showed complete absence of endogenous OA1 protein, thus representing a suitable model for setting up an efficient gene transfer procedure. To correct the genetic defect in these cells, we used a retroviral vector carrying the OA1 cDNA and exploited a melanocyte-keratinocyte coculturing approach. Despite their lower replication rate with respect to wildtype cells, the patient melanocytes were efficiently transduced and readily selected in vitro, and were found to express, process, and properly sort large amounts of recombinant OA1 protein. These results indicate the feasibility of efficiently and stably transducing in vitro not only normal neonatal, but also mutant adult, human melanocytes with nonmitogenic genes.

Signaling pathways in melanosome biogenesis and pathology

“…The macromelanosomal phenotype suggests a defect in melanosome biogenesis, however it also displays variable expressivity in vivo (Garner and Jay, 1980; O’Donnell et al, 1976; Schnur et al, 1998; Wong et al, 1983) and in vitro , since giant melanosomes were observed in melanocytes obtained from Oa1 -KO mice, but not human patients (Palmisano et al, 2008; Schiaffino et al, 2002). It is possible that in different pathological conditions the deficiency of OA1 manifests with alternative phenotypes, which could nevertheless provide insights into the function of the receptor.…”

“…It is possible that in different pathological conditions the deficiency of OA1 manifests with alternative phenotypes, which could nevertheless provide insights into the function of the receptor. For instance, primary cultures of human melanocytes from patients with ocular albinism showed no macromelanosomes, but a striking prevalence of mature over immature melanosomes, compared to wild-type or OA1-transduced cells (Schiaffino et al, 2002). Similarly, transient OA1 downregulation in MNT1 cells did not result in the formation of classical macromelanosomes (Giordano et al, 2009).…”

An unconventional dileucine-based motif and a novel cytosolic motif are required for the lysosomal and melanosomal targeting of OA1

“…This time interval allows accumulation of the tracer into the lateendocytic pathway, particularly into lysosomes, because, in these chase conditions, its staining overlaps extensively with DAMP and LAMP1 (data not shown). In melanocytes, the endogenous OA1 has been localized to late endosomes and lysosomes (Samaraweera et al, 2001) and to melanosomes (Schiaffino et al, 1996;Schiaffino et al, 1999;Schiaffino et al, 2002;Basrur et al, 2003); however, quantitative studies with multiple markers have never been performed.…”

New mutations identified in the ocular albinism type 1 gene

“…This applies especially to gene therapy approaches that might be able to deliver the wild-type OA1 protein to the RPE of the affected patients, thus improving in general their visual acuity by enhancing foveal differentiation. Indeed, although essentially an issue for future applications, this has already being tested in an in-vitro gene therapy approach with cultured melanocytes from ocular-albinism patients, where the phenotype has been rescued using infection with a retrovirus carrying the wild-type OA1 protein (Schiaffino et al, 2002).…”